): Cancer development is associated with changes in the mechanical properties of cells as well as the extracellular matrix. Few facts are known about the interplay of cells and the extracellular matrix (ECM) on the molecular level in respect to the transformation from a normal to a malignant cell. The objective of this proposed project is to develop a radically new approach to investigate quantitatively the mechanical properties of cells and ECM and to correlate those with signaling pathways. Cells will be embedded in true 3D-matrixes and a method based on the combination of atomic force microscopy (AFM) and the optical microscopy techniques, laser scanning confocal, wide-field fluorescence, and phase contrast microscopy will be applied to extract mechanical volume properties ofthe cells and the surrounding matrix with nanometer resolution. Because ofthe heterogeneous constitution, on the nanometer scale, ofthe cells as well as the matrix, new analysis methods based on finite element analysis have to be developed and applied in order to extract quantitative data to create 3D-elasticity maps ofthe samples. Using this novel method, the influence of local induced mechanical stress on the shape, surface morphology, and the signaling pathways ofthe cells will be investigated. In general, this ansatz enables us to correlate the physical properties, malignant transformations, and molecular tumorigenic signaling pathways. The proposed technique is a single cell method, which allows for the exploration of inhomogeneities in a cell population. The specific aims of this proposed project are: 1) Development of mechanical nanotomography of cells embedded in true 3D-matrixes for quantitative analysis of local mechanical properties. 2) Development of gel encapsulation of living cells compatible with mechanical nanotomography. The cell response to encapsulation in 3D-matrixes of different stiffness and composition will be measured. This method will be applied to cells embedded in matrixes mimicking ECM. 3) Development of a method based on combined AFM/optical microscopy to probe the response ofthe embedded cells to external mechanical stimuli.